ARTICLE https://doi.org/10.1038/s42003-021-01857-0 OPEN Creb5 establishes the competence for Prg4 expression in articular cartilage Cheng-Hai Zhang1, Yao Gao1, Unmesh Jadhav2,3, Han-Hwa Hung4, Kristina M. Holton5, Alan J. Grodzinsky4, ✉ Ramesh A. Shivdasani 2,3,6 & Andrew B. Lassar 1 A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the devel- opment of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes 1234567890():,; derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage. 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute at Harvard Medical School, Boston, MA, USA. 2 Department of Medical Oncology and Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA. 3 Departments of Medicine, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA, USA. 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 5 Research Computing, Harvard Medical School, Boston, MA, USA. 6 Harvard Stem Cell Institute, Cambridge, MA, USA. ✉ email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:332 | https://doi.org/10.1038/s42003-021-01857-0 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-01857-0 steoarthritis (OA) affects over 30 million US adults (CDC required to activate Prg4 expression, in response to TGF-β and Ostatistics). Interventions that block the progression of OA EGFR signaling. are currently unknown. With an eye toward this goal, we have sought to develop a comprehensive understanding of the regulatory network that regulates the differentiation and main- Results tenance of articular cartilage, which plays a central role in Creb5, a TF expressed selectively in Prg4+ chondrocytes.In maintaining the low-friction environment of the joint space. A newborn bovine knee joint cartilage, superficial zone chon- hallmark of cells comprising the articular cartilage is their drocytes (SZCs) expressing Prg4/lubricin are readily distinguished expression of proteoglycans, such as the protein lubricin, encoded from Prg4/lubricin-negative deep zone chondrocytes (DZCs, by the Prg4 gene, that lubricates the joint and protects against the Fig. 1a). As the profound differences between these two tissues development of OA1,2. Prg4 is specifically expressed in the likely have a transcriptional basis, we employed RNA-Seq to superficial-most layer of the articular cartilage, but not by deeper identify SZC-specific TF genes. Reflecting their common devel- layers of this tissue2–7. Fate mapping studies have established that opmental origin8–10, SZCs and DZCs differed by only 320 genes Prg4-expressing cells in embryonic and early postnatal joints (Supplementary Data 1; false discovery rate, FDR < 0.05), constitute a progenitor pool for all regions of the articular car- including ~67-fold higher Prg4 mRNA levels in SZCs, as expec- tilage in the adult8–10. These findings are consistent with prior ted, and >15-fold higher levels of other SZC marker genes such as studies indicating that both superficial and deep zones of the Vitrin, Epha3, Wif1, and Thbs4 (Supplementary Data 1; Fig. 1b). articular cartilage (plus other synovial joint tissues) specifically Notably, we found that 29 transcriptional regulators were more arise from Gdf5-expressing cells in the embryo11–13. In both highly expressed (at least twofold) in SZCs than in DCZs; and humans and mice lacking Prg4, the surface of the articular car- only one TF (Sall1) was more highly expressed in DZCs (Table 1). tilage becomes damaged and precocious joint failure occurs1,2. The TF gene Creb5 was the most differentially expressed tran- Notably, decreased levels of lubricin have been observed in the scriptional regulator, with ~25-fold higher levels in SZCs than in synovial fluid following either surgically induced OA in sheep14, DZCs (as determined by RNA-Seq; Fig. 1c), approximately equal in human synovial fluid samples from patients with either OA or to the differential expression of Prg4 in these tissues. Because rheumatoid arthritis15, and in the menisci from OA patients16. TGF-β induces Prg4 specifically in SZCs in vitro36 and maintains Furthermore, a decrease in Prg4/lubricin expression during Prg4 expression in articular cartilage in vivo37, we assessed aging17 correlates with increasing sensitivity of aged knees to mRNA levels in SZCs and DZCs cultured with or without TGF- cartilage degradation following knee joint destabilization18. Most β2. RT-qPCR revealed approximately tenfold induction of Prg4 notably, loss of lubricin (in Prg4−/− mice) has been noted to by TGF-β2 only in SZCs, as others have reported36; and a result in significantly higher levels of peroxynitrite, superoxide, selective 250-fold (Prg4) and 40-fold (Creb5) greater expression of and cleaved caspase 319, which correlates with increased levels of these transcripts, in response to TGF-β2, in SZCs versus DZCs both whole-joint friction and cellular apoptosis in Prg4−/− mice (Fig. 2a, compare lanes 2 and 4). compared with either wild-type or Prg4+/− mice20. Indeed, a The basic-leucine zipper (bZIP) DNA-binding domain of hallmark of both aging21,22 and OA23 is a loss of cells in the Creb5 shares high sequence homology with those of Atf240 and superficial zone of the articular cartilage. Conversely, if lubricin Atf741. In addition, all three TFs carry two conserved N-terminal protein is either injected directly into the synovial fluid24–28 or Threonine/Proline residues (T59 and T61 in Creb5) that are overexpressed in the knee joint (via transgene or AAV;29,30) the substrates for the P38, Jun N-terminal (JNK), and extracellular articular cartilage tissue is protected from degradation following signal-regulated (ERK) kinases (reviewed in42,43). Consistent surgically induced joint destabilization. Taken together, these with selective Creb5 mRNA expression in SZCs, a ~65-kDa findings indicate that Prg4/lubricin counters the signaling path- protein recognized by both Creb5 and phospho-specific Creb5 ways that lead to cartilage destruction and suggest that identifying (T61) antibodies is enriched specifically in SZCs (Fig. 2b). a means to induce the sustained expression of Prg4 in the Infection of SZCs with lentivirus encoding an shRNA directed articular cartilage may attenuate the degradation of articular against the 3′UTR of Creb5 substantially diminished these protein cartilage observed during either aging or OA. levels (Fig. 3c). Of note, phospho-P38 kinase, the active form that While Foxo31, Nfat32, and Creb133 transcription factors (TFs) can phosphorylate Creb5 on T61, is also more abundant in SZCs have been found to modulate Prg4 expression in articular chon- than in DZCs (Fig. 2b). Although TGF-β2 diminished the level of drocytes, the TFs that drive either tissue-specific expression of Creb5 mRNA to ~30% of that in untreated SZCs (Fig. 2a, lanes 1 Prg4 or region-specific expression of this gene in the superficial and 2), this decrease had no appreciable effect on either total or zone of articular cartilage have not yet been elucidated. In addi- phospho-Creb5 protein levels (Figs. 2b and 3c). Consistent with tion, as Prg4 is expressed in articular chondrocytes but not in the restricted expression of Creb5 in the superficial zone of bovine growth plate chondrocytes, we speculated that identification of articular cartilage, we similarly detected nuclear-localized the TFs that control expression of this gene in the superficial zone Creb5 specifically in the lubricin-expressing superficial zone of of articular cartilage may elucidate how these distinct chon- articular cartilage in adult human femoral head tissue (Fig. 2c). drocyte cell fates are regulated. Several signaling pathways, Prg4 is initially expressed in the articular perichondrium, including Wnt11,34,35, TGF-β 36,37, and EGFR38, have all been which encases the epiphyses of the developing long bones found necessary to maintain the expression of Prg4 in the (depicted schematically in Fig. 2d); and is subsequently expressed superficial zone of articular cartilage. Interestingly, however, in the superficial-most layer of mature articular cartilage, but not injurious mechanical compression39, TGF-β1 exposure36, and by deeper layers of this tissue2–7. We used RNA in situ shear stress from fluid flow33 can induce Prg4 expression exclu- hybridization to localize Creb5 transcripts in relation to Prg4 sively in explants taken from the superficial zone of bovine and Collagen 2a1 (Col2a1) in the elbow joints of newborn mice. articular cartilage, and not in explants from middle or deep zones. Consistent with our findings in bovine articular chondrocytes, we This highly restricted induction implies that superficial